Although cholesterol levels are widely regarded as a reliable indicator of prospective health problems attributable, for example, to coronary disease, these levels are not easily measured by routine analytical methods involving spectrophotometry since cholesterol is not a colored material. Thus previously known methods for cholesterol determination generally require a color derivatization step. Examples of these include the Liebermann-Burchard reaction, the Zak reaction and the Abell-Kendall reaction, all of which occur in non-aqueous media and require special control of reaction conditions. Another reaction known in this regard is the oxidizing enzyme-dye reaction.
The color derivatization steps known heretofore, however, do not provide a derivative of the cholesterol molecule itself; rather, they provide secondary products of cholesterol oxidation which means that the spectrophotometric measurement of cholesterol is an indirect one. That is, the intensity of the color is not a direct measure of cholesterol concentration.
As noted hereinbefore, most routine analytical methods for practical purposes employ spectrophotometry. Spectrophotometry refers to the measurement of the absorption or transmission of incident light through solutions of test compounds. Typically, compounds of interest have characteristic spectra, transmitting or absorbing specific wavelengths of light, which can be used to determine the presence of these compounds or measure their concentration in test samples. Instruments designed for spectrophotometric absorption have a light source, for which the emitted wavelength is known and may be adjusted, and one or more detectors sensitive to desired wavelengths of transmitted or reflected light. Spectrophotometric absorption can be used to determine the amount of a given compound that is present in a test sample.
Circular dichroism (CD) is a special type of absorption method in which the molecular composition of an analyte results in differential absorption of incident light not only at a specific wavelength but also of a particular polarization state. Circular dichroism is a chiroptical method which allows one to differentiate between different enantiomers; that is, optical isomers having one or more asymmetric carbon atom (chiral) centers. When utilizing CD, generally a sample is illuminated by two circularly polarized beams of light traveling in unison. Both beams pass through the sample simultaneously and are absorbed. If the sample is optically active, the beams are absorbed to different extents. The differences in absorption of the beams can then be displayed as a function of the wavelength of the incident light beam as a CD spectrum. No difference in absorption is observed for optically inactive absorbers so that these compounds are not detected by a CD detecting system. The use of CD as a chiroptical method has been fully described in scientific literature, such as Lambert, J. B. et al. "Organic Structural Analysis", Macmillan, New York, N.Y. 1976.
Early applications of the CD method dealt primarily dealt with elucidation of molecular structures, especially natural products for which a technique capable of confirming or establishing absolute stereochemistry was critical. However, CD has also reportedly been used in a clinical method to quantitatively determine unconjugated bilirubin in blood plasma, Grahnen, A. et al. Clinica Chimica Acta, 52, 187-196 (1974). In the method thus disclosed, a complex was formed between bilirubin and human serum albumin as a CD probe for bilirubin analysis.
Clinical applications of circular dichroism are also discussed by Neil Purdie and Kathy A. Swallows in Analytical Chemistry, Vol. 61, No. 2, pp. 77A-89A (1989), herein incorporated by reference. Possible clinical applications of CD are disclosed to include measurement of cholesterol levels and detection of anabolic steroids. However, suitable chemical reagents for carrying out such testing are not disclosed.
Regarding the use of spectrophotometric absorption, fluorescence, derivative spectrophotometry or CD methods herein disclosed to measure cholesterol levels, it is noted that the population at large is continually advised that it is prudent to know serum cholesterol levels and constantly reminded that an uncontrolled diet and a lack of exercise can lead to accumulation of arterial plaque that will increase the risk of atherosclerosis and coronary heart disease. Statistical studies, such as those reported by Kannel, W. B. et al. in "Serum Cholesterol, Lipoproteins and the Risk of Coronary Heart Disease: The Framingham Study" Ann. Intern. Med., 74:1-11 (1971) and Castelli, W. P. et al. in "Incidence of Coronary Heart Disease and Lipoprotein Cholesterol Levels", JAMA, 256:2835-2838 (1986), have shown that other risk factors, such as age, gender, heredity, tobacco, alcohol consumption etc. must also be considered when counselling patients about the risks.
The magnitude of the program for screening the general public is so immense that automated methods for cholesterol determinations are necessary. The tests currently used differ in complexity from the simple dip-stick approach, which uses a color sensitive reaction on a paper support, to sophisticated lipid profile tests in which the distribution of cholesterol among the various solubilizing molecular species is determined, Abbott, R. D. et al. "Joint Distribution of Lipoprotein Cholesterol Classes, The Framingham Study, Arteriosclerosis, 3:260-272 (1983). Here, the dip-stick approach is only a preliminary qualitative test upon which a decision for the fuller, more quantitative measurement can be based.
At the conclusion of a recent extensive study of how health risk factors are related to elevated levels of serum cholesterol, a report entitled "Current Status of Blood Cholesterol Measurement in Clinical Laboratories of the United States, A Report from the Laboratory Standardization Panel of the National Cholesterol Education Program", Clin. Chem., 34:193-201 (1988), was prepared by the Laboratory Standardization Panel (LSP) of the National Cholesterol Education Program (NCEP). In this report, the measure or risk was correlated with three ranges of total cholesterol (TC): low risk if less than 200 mg/dL; moderate risk in the range 200-239 mg/dL; and high risk if greater than 240 mg/dL. In order to place a particular individual into one or another of these categories, all that is required is a serum TC measurement. The other risk factors, such as those identified by Kannel et al. and Castelli et al., supra, are then added as a basis for further patient counselling. This relatively simple approach replaces an earlier recommendation in Kannel et al., supra, and in Superko, H. R. et al. "High-Density Lipoprotein Cholesterol Measurements--A Help or Hinderance in Practical Clinical Medicine", JAMA 256:2714-2717 (1986) in which relative risk was established using a ratio of TC to high density lipoprotein cholesterol (HDL-C) equal to 5. A ratio lower than 5 implies a high level of HDL-C and a low relative risk. For this diagnosis, HDL-C is measured in a second independent test.
The same report by the LSP hastened to add that there were serious inaccuracies in measurements made by numerous clinical laboratories in the determination of the amount of TC present in human serum reference standards.
Statistically the results showed that, in data from 1500 laboratories, 47% failed to measure the true value to within a coefficient of variance (CV) of .+-.5%, and 18% of these failed at a CV of .+-.10%. As a consequence, the LSP recommended that an improvement in CV to within .+-.3% for TC should be achieved by 1992. Recent surveys indicate that certified laboratories are well on their way to meeting that challenge, using the current clinical methods and instrumentation, as reported, e.g. in Posnick, L. "Labs now Better at Cholesterol Tests Data Show", Clin Chem News 15(9):14 (1989). The LSP did not report the inaccuracies associated with the determination of the distribution of cholesterol among the various lipids and lipoproteins, but did indicate that an evaluation would be made in the future. The very poor proficiency and lack of reliability in the measurement of serum or plasma HDL-C, has been eloquently described in Superko, H. R. et al., supra, and in Warnick, G. R. et al. "HDL Cholesterol: Results of Interlaboratory Proficiency Test" Clin. Chem. 26:169-170 (1980) and Grundy, S. M. et al. "The Place of HDL in Cholesterol Management. A Perspective from the National Cholesterol Education Program" Arch. Inter. Med. 149:505-510 (1989) where interlaboratory CV's as high as 38% were reported. A 1987 evaluation by the College of American Pathologists (CAP) of the measurement of the same sample for HDL-C by over two thousand laboratories showed that more than one third differed by more than 5% from the reference value. Interlaboratory CV's amount groups using the same method did improve to 16.5%, but it is still too imprecise to be of any predictive clinical value. For this reason, the TC:HDL-C ratio is no longer used in risk assessment, although it offers potential advantages in defining the true clinical picture.
Regarding presently used lipid profile studies, cholesterol is known to be distributed in the serum mainly associated with high density lipoprotein (HDL-C) and low density lipoprotein (LDL-C) fractions and with triglycerides as the very low density lipoprotein cholesterol (VLDL-C) fraction. There is plenty of statistical evidence from a number of long term clinical tests to indicate that a high proportion of HDL-C and a low proportion of LDL-C is associated with lower relative risk or in simpler terms, high levels of LDL-C are to be avoided where possible. HDL-C is beneficial, provided the level is not excessively low, i.e., less than 30 mg/dL. VLDL-C cholesterol has not been implicated in any risk determination, but high triglyceride itself can be a serious problem.
In a typical lipid profile study, total cholesterols are measured directly and HDL-C is measured in the supernatant remaining after treatment of the sample with an agent to precipitate out LDL-C and VLDL-C. VLDL-C is taken to be a fixed fraction (e.g., 0.2) of the triglyceride, which is also measured directly in a separate assay. LDL-C is calculated from these figures and is not measured directly. The propagation of errors in each of the three independent measurements make LDL-C the fraction known with least overall accuracy and precision, although it may be the most significant aspect of cardiovascular risk. Because of this, it is difficult to meaningfully monitor and establish that clinical progress has been made in LDL-C reduction therapy with time.
At a workshop and subsequent roundtable session held at the 43rd Meeting of the American Association for Clinical Chemistry, the present state of the art in this area was summarized, as reported in Baillie, E. G. et al. "Standardization and Clinical Utility of Lipid Determinations", Workshop, 43rd National Meeting American Association for Clinical Chemistry, 1991 and Warnick, G. R. "Standardization of HDL Cholesterol Measurement" Roundtable, 43rd National Meeting, American Association for Clinical Chemistry, 1991. From these proceedings, it was concluded that accuracy is essential in HDL-C measurement. While presently available precipitation methods can give satisfactory results, the values obtained by these methods in routine clinical laboratory settings do not meet real medical needs. The CAP comprehensive chemistry proficiency survey from 1982 to 1991 for HDL-C showed interlaboratory CVs of about 20% in 1991, with no overall improvement since 1982. The CVs delivered by clinical instruments used for HDL-C measurements ranged from 7.6% for the DIMENSION to 50% for the EKTACHEM.
At the sessions, it was also noted that direct methods for LDL cholesterol are needed. The use of triglyceride determinations to estimate VLDL-C by the Friedewald equation is, at present, the method of choice. To quote the workshop syllabus, "The variability typically observed in the measurement of total and HDL cholesterol and triglycerides may preclude attaining acceptable precision. In fact, to achieve the ideal precision in LDL cholesterol estimation, the precision of the constituent measurements must be better than their ideal specifications."
It is thus established that there is a need for a relatively simple, reliable and repeatable assay method to directly and simultaneously determine the amount of cholesterol, both total cholesterol and its distribution among the various subfractions without the need for precipitation or separate measurements of these subfractions.